[Show abstract][Hide abstract] ABSTRACT: The technical limitations of isolating target cells have restricted the utility of pluripotent embryonic stem (ES) cells. For example, early cardiac (i.e., precontractile) cells have not been isolated from ES cells. Here, we find that direct expression of reporter genes under cell-specific promoters-the currently available strategy for isolating cells lacking cell-specific surface markers-is ineffective for isolating progenitor cells. This was due to the weak activity of cell-specific promoters, particularly in ES cells at early stages. We show that adenoviral conditional targeting efficiently isolates viable ES cell-derived target cells without harmful effects. In this strategy, we employ the alpha-myosin heavy chain and Nkx2.5 promoter to visualize and purify efficiently differentiated and primitive cells of the cardiac lineage, respectively. While the former cells predominantly expressed sarcomeric proteins and maintained contractile function, the latter demonstrated neither of these features, but rather exhibited expression patterns characteristic of a mixture of primitive cells and cardiomyocytes. Interestingly, smooth muscle actin was predominantly expressed in the latter cells, and both functionally known and unknown genes were systematically identified, demonstrating the benefits of this system. Thus, our method facilitates molecular and cellular studies of development and ES cell-derived cell therapy.
[Show abstract][Hide abstract] ABSTRACT: Despite the enormous potential of conditionally replicating adenoviruses (CRAs), the time-consuming and laborious methods required to construct CRAs have hampered both the development of CRAs that can specifically target tumors with multiple factors (m-CRA) and the efficient analysis of diverse candidate CRAs. Here, we present a novel method for efficiently constructing diverse m-CRAs. Elements involving viral replication, therapeutic genes, and adenoviral backbones were separately introduced into three plasmids of P1, P2, and P3, respectively, which comprised different antibiotic resistant genes, different ori, and a single loxP (H) sequence. Independently constructed plasmids were combined at 100% accuracy by transformation with originally prepared Cre and specific antibiotics in specific Escherichia coli; transfection of the resulting P1+2+3 plasmids into 293 cells efficiently generated m-CRAs. Moreover, the simultaneous generation of diverse m-CRAs was achieved at 100% accuracy by handling diverse types of P1+2 and P3. Alternatively, co-transfection of P1+3 and P2 plasmids into Cre-expressing 293 cells directly generated m-CRA with therapeutic genes. Thus, our three-plasmid system, which allows unrestricted construction and efficient fusion of individual elements, should expedite the process of generating, modifying, and testing diverse m-CRAs for the development of the ideal m-CRA for tumor therapy.